A group of growing dogs supplemented with cholecalciferol (vitamin D₃; HVitD) was studied vs. a control group (CVitD; 54,000 vs. 470 IU vitamin D₃/kg diet, respectively) from 3 to 21 wk of age. There were no differences in plasma levels of P_i and growth-regulating hormones between groups and no signs of vitamin D₃ intoxication in HVitD. For the duration of the study in HVitD vs. CVitD, plasma 25-hydroxycholecalciferol levels increased 30- to 75-fold; plasma 24,25-dihydroxycholecalciferol levels increased 12- to 16-fold and were accompanied by increased renal 24-hydroxylase gene expression, indicating increased renal 24-hydroxylase activity. Although the synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] was increased in HVitD vs. CVitD (demonstrated by [³H]1,25(OH)₂D₃ and increased renal 1-hydroxylase gene expression), plasma 1,25(OH)₂D₃ levels decreased by 40% as a result of the even more increased metabolic clearance of 1,25(OH)₂D₃ (demonstrated by [³H]1,25(OH)₂D₃ and increased gene expression of intestinal and renal 24-hydroxylase). A shift of the Ca set point for parathyroid hormone to the left indicated increased sensitivity of the chief cells. Effective counterbalance was provided by hypoparathyroidism, hypercalcitoninism, and the key regulator 24-hydroxylase, preventing the development of vitamin D₃ toxicosis.